Electrophoresis is used for the separation of ionic components in a sample solution. In the prior art, a sample solution is placed on or in a supporting medium (e.g. a piece of paper or block of gel) and a voltage is applied across the supporting medium. The charged components migrate through the supporting medium with a speed that is related to the charge on each component, the effective size of the component, and the applied voltage so that, over a given period of time, the components move different distances through the medium away from their common starting points.
The electrophoretic separation of biological compounds such as proteins or nucleic acids has become a widely used method for the identification and purification of individual components in a sample mixture. Present gel electrophoresis techniques require the use of voltages generally less than about 100 volts for time periods greater than about 30 minutes unless special and relatively expensive gels are employed. Even when using special gels, the time periods are greater than about 30 minutes.
Many biological compounds of interest consist of one component, such as a protein, bound to another component, such as a nucleic acid. Some of these compounds are bound together tenuously when removed from their natural biological systems and they may fall apart before the time required for the completion of the separation, purification and/or identification of the components using the present techniques of gel electrophoresis (i.e., greater than about 30 minutes).
The time required for the electrophoretic separation may be decreased by increasing the voltages used in the gel electrophoresis. Unfortunately, increasing the voltages also leads to an increase in the temperatures generated during separation. This increase in temperature may degrade the quality of the electrophoresis in at least two ways. First, since the medium used in gel electrophoresis may melt near or slightly above room temperature, gel electrophoresis techniques of the prior art at elevated voltages (i.e., greater than about 100V) may produce areas of the gel that may exceed the melting point of the gel. A sample loaded into a gel medium, such as an agarose gel, may become lost if parts of the gel become liquid and can no longer contain the sample.
Second, biological components, such as polypeptides, nucleic acids or other macromolecules may denature or significantly degrade at the elevated temperatures encountered when using high voltages for gel electrophoresis. Some of the denatured material may be renatured, but degraded components cannot, in general, be returned to their natural state for study or use. Both of these limitations to present gel electrophoresis techniques often limit the voltage to below about 100 V and the time periods of the separations to greater than about 30 minutes.
FIG. 2 shows a graphical representation of time and voltage conditions for conducting agarose gel electrophoresis. The area labeled 6 represents the conditions available for agarose gel electrophoresis using the techniques of the prior art. In general, the voltage is limited to less than 100 V and the time required to complete the separation exceeds 30 minutes.
Even when the gel electrophoresis is conducted within the limitations of the prior art techniques, the practitioner may still encounter problems with undesired temperature variations within the gel. During the course of preparation of the gel medium, it is possible that there may be areas of inhomogeneity in the final gel due to incomplete mixing of the gel material. While the electrophoresis is in progress, these areas of inhomogeneity may experience temperatures above that of the gel as a whole. Those areas of the gel may even melt. But, even if the temperature variation is not enough to melt the gel, the components of the sample solution may be exposed to temperatures that lead to denaturation and/or degradation. In either case, the sample components may not be recoverable or those recovered may not be the ones expected. It would be of great practical value to be able to continuously and conveniently monitor the temperature at various points throughout the gel. The prior art gel electrophoresis techniques do not provide the means necessary to do that.
The insufficiencies of the prior art techniques of gel electrophoresis limit the types of separations that may be performed. In addition, there may be some uncertainty with regard to the nature of the recovered materials when a separation is conducted at or near the limits of the present techniques.
Therefore, it is an object of the present invention to provide a method for rapidly and efficiently separating the components of a sample solution by gel electrophoresis.
It is another object of the present invention to provide an apparatus for rapid electrophoretic separation of the components of a sample solution.
It is another object to provide for enhanced resolution of the separation of sample components when subjected to a gel electrophoresis procedure.
It is a further object of the present invention to provide a means for monitoring the temperature of the mediums containing the sample before, after and/or during the completion of the electrophoretic separation.